Ovarian tissue undergoes marked morphological and functional changes during the reproductive cycle. These changes require the availability of cholesterol for membrane synthesis and steroidogenesis. However, the sources of cholesterol in the various ovarian components and the structural organization of the pathways by which it is obtained have not been clearly defined. Our first objective is to develop a cytochemical procedure to localize at the electron microscope level 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in the de novo synthesis of sterol, dolichol and ubiquinone. Cell fractionation studies indicate that this enzyme is present in ovaries but it is not clear which compartments of this organ express the enzyme. It is also not resolved which organelle(s) (rough or smooth ER, or mitochondria) bear this enzyme. Information on the cellular and subcellular distribution of this enzyme would provide cluse as to control of its activity. Our second objective is to examine the relationships between plasma lipoproteins, which are exopenous sources of cholesterol, and ovarian cells. The main goals are to: 1) identify which cell types in the ovary accumulate high and low density lipoproteins (HDL, LDL), 2) localize and characterize the initial sites at which these lipoproteins bind, and 3) determine if HDL and LDL are internalized and delivered to specific organelles for processing. This will be accomplished by a morphological and biochemical analysis of the perfused ovary in situ and of luteinized granulosa cells in culture. Presentation of liporpoteins by vascular perfusion simulates in vivo conditions. This allows an analysis of lipoprotein metabolism in a system that contains all cellular components in their normal structural relationships, in contrast to the study of a single cell type in culture. The uptake and metabolism of HDL and LDL labeled with 12 5I, ferritin or collodial gold and HDL and LDL bearing 14C-cholesteryl oleate will be examined in both systems. Morphological probes will identify the sites of uptake and the pathwayof intracellular processing of HDL and LDL. Biochemical studies will provide complementary information on the metabolism of these lipoproteins. The structural and functional data from these studies should clarify the roles of de novo sterol synthesis and utilization of lipoprotei-carried cholesterol in ovarian cells at different phases of function.